HIV-1 causes the largest number of deaths from a single infectious agent in the world today. Despite considerable advances in treatment of HIV-1 infection and AIDS, which it causes, there is an urgent need for better methods of prevention and treatment of HIV-1 infection. The development of a safe and effective vaccine to prevent HIV-1 infection or the subsequent development of AIDS is therefore of the utmost importance. This effort, however, has been hampered by a lack of understanding of the correlates of protective immunity and a lack of the tools needed to measure effective anti-HIV-1 immune responses. We have developed a Multi-Clade HIV-1 Proteomic Chip (MC-HIV-1 chip) that can be used as a tool to rapidly screen antibody responses to HIV-1 elicited in vivo in response to infection or vaccination. The current version of the MC-HIV-1 chip which is available commercially from Antigen Discovery Incorporated (ADI) expresses over 100 HIV-1 proteins, protein fragments and epitopes from clades A1, A2, B, C and D which together comprise 74% of HIV-1 infections worldwide (Stephens 2012). Our preliminary studies show that the MC-HIV-1 chip can be used to identify the specificity of HIV-1 antibodies, track changes in the humoral immune response to HIV-1 during infection and may also be able to identify the clade of an HIV-1 infection. In this application we propose to improve the MC-HIV-1 chip by expanding it's coverage of HIV-1 subtypes, circulating recombinant forms (CRF), disulfide bound antigens, glycosylated antigens, epitopes and HIV-2 groups A and B thereby creating a Pan-HIV Proteomic Chip. We further propose to validate the MC-HIV chip and Pan HIV chip with a variety of sera and antibodies. We hypothesize that the improved Pan-HIV Chip will allow identification of the type, group and subtype of an HIV infection, will facilitate rapid characterization of humoral immune response to nearly all HIV infections and vaccination regimens, will identify reactivity with some broadly neutralizing epitopes and will differentiate natural from vaccine-induced humoral immunity. We have the following aims: 1 - We will expand the MC-HIV-1 chip to include HIV-1 subtype G, CRF01 AE and CRF01 AG as well as HIV-2 groups A and B, thereby creating a Pan-HIV chip. This will expand coverage to > 90% of HIV-1 infections and virtually all HIV-2 infections globally. 2 - We will improve the Pan-HIV chip by including disulfide bridges and glycosyl groups in the external proteins, gp120 and gp41, and in their fragments, including the variable loops of gp120. We will also include consensus sequences of key epitopes that differentiate clades as well as those that are the targets of braodly neutralizing (BN) antibodies. This will improve reactivity with BN and clade specific antibodies as well as antibodies that recognize conformational and glycosyl epitopes.3 - We will assay the ability of the current MC-HIV-1 chip as well as the improved Pan-HIV chip to react with patient sera from progressors, non-progressors, from early vs. late in infection, from patients infected with different types, groups and clades of HIV and following vaccination. We will also assay reactivity of all classes of broadly neutralizing monoclonal antibodies with the current and improved HIV chips. This aim will demonstrate the utility of the current MC-HIV-1 chip and improved Pan-HIV chip in vaccine research and in characterizing the humoral immune response to HIV infection. Future directions will include screening vaccinee sera and optimizing the Pan HIV chip for differentiating humoral immune responses to vaccinination from responses to natural infection.